Background: Ubiquitin mediated degradation and trafficking is critical for protein quality control in eukaryotic cells. Significant efforts by man investigators have elucidated many mechanistic details of E1-E2-E3 ubiquitin transfer cascades. However, the structural mechanism by which a fourth type of E4 ligase catalyzes polyubiquitin chain elongation is unknown. Furthermore, unpublished data from the co-sponsor's laboratory has implicated that an interaction between the human E4, UbE4B, and a ubiquitinated protein processing enzyme, VCP, is critical to the degenerative phenotype observed in the disease multi-system proteinopathy (MSP). Despite its role in MSP, cancer, and other neurodegenerative processes, the pathways and protein substrates regulated by UBE4B-mediated polyubiquitination remain elusive. Long-term Objectives: The long-term goals of the PI are to understand how the processing of polyubiquitination targets contributes to cellular homeostasis, particularly in pathways mediated by UBE4B and VCP. Specific Aims: This proposal aims to (1) Determine the detailed molecular basis for the yeast E4's activity and (2) Elucidate mechanisms and pathways of E4 from higher eukaryotes. Experimental Design: In aim 1 this proposal will elucidate the structural mechanism of the better-characterized yeast E4 enzyme, Ufd2p. In order to facilitate this aim, both x-ray crystallography and cyro-electron microscopy will be used to obtain a structure of catalytic intermediate of the E4 elongating a Ub chain in complex with an E2 bound to a donor Ub. Quantitative binding assays and ubiquitin transfer assays will then be used to further test hypotheses inferred from structural data on the mechanism of E4 polyubiquitination. In aim 2, this proposal will use a multi-faceted approach involving genetic screening in a Drosophila model of MSP, quantitative biochemical assays and proteomics to identify the E2 and E3 enzymes and ultimately the protein substrates that comprise the pathways regulated by UbE4B and VCP. Relevence to health: The proposed research will provide new insight into the mechanism of protein trafficking and degradation mediated by VCP and UBE4B, and thus facilitate further development of therapy for cancer, MSP and related diseases using either or both proteins as targets or prognostic indicators for treatment.